The APOBEC3 family comprises seven different proteins (APOBEC3A [A3A] to A3H), which may act as strong mutators of proviral genomes. The accessory HIV protein Vif counteracts the antiviral activity of some but not all APOBEC3 variants. Although the global HIV-1 pandemic is driven by non-B subtypes, in particular subtype C, most data concerning the mode of action of Vif have been derived from subtype B molecular clones in conjunction with A3G. APOBEC3 molecules, however, differ not only in catalytic activity and expression but also in susceptibility to HIV-1 Vif mediated degradation. We hypothesize that broad spectrum Vif activity is the driving force for transmission/establishment of infection while suboptimal Vif activity is essential for emergence of escape variants and diversification in the later stages of disease. This hypothesis will be tested in three complementary specific aims using established and new assays. We will perform a comprehensive analysis of the function of pandemic, non-pandemic and patient- derived Vif alleles (Aim 1), dissect the relationship between Vif evolution and skewed proviral archives in patients (Aim 2), and determine the plasticity of Vif in forced selection experiments by passaging HIV in the presence of increasing APOBEC3 expression (Aim 2). Lastly, we will establish which APOBEC3 restricts viral spread of CXCR4 and CCR5 using viruses encoding mutant Vif variants in a humanized mouse model (Aim 3). These experiments will collectively establish the putative role of Vif as virulence factor, create genotype to phenotype assays to predict Vif activity in a subtype specific context and identify specific APOBEC3 proteins relevant for restriction and diversification in vivo. This knowledge will help in the discovery of drugs targeting this diverse viral-host interface and improve the already available treatment options by identifying patients at high risk for viral escape. PUBLIC HEALTH RELEVANCE: This proposal will investigate to what extent Vif alleles representative of all HIV subtypes and Vif variants derived directly from infected individuals differ in their anti-APOBEC3 activities and impact on viral diversification. These findings will allow for differential personalized treatment strategies adapted to the individual's own APOBEC3 repertoire.